0.25% (wt) VA-044 (thermally-triggered initiator) (This one can be hard to find; we source ours from Wako Australasia.)

0.05% (wt/vol) of saponin (This is useful if you want to increase specimen permeability, or if the specimen is large.)

Mix all ingredients together. Ingredients should be kept on ice during preparation of the solution to prevent premature polymerisation.

Procedures:

(1) Incubation

Immerse specimens in hydrogel solution for 1-2 weeks at 4° C. Larger specimens should be immersed for longer, and specimen type may be important too (e.g., lizards probably take longer than tadpoles). Users should be aware that the immersion time here is longer than for the original STABILITY protocol (Wong et al., 2013). This may be due to longer fixation in formalin, which seems to use up some biomolecule binding sites.

(2) Curing

Any type of vegetable/nut/canola oil can be used for curing (approximately 3 ml). Carefully pour the oil on the surface of the hydrogel solution to form an airtight layer. Some researchers use a vacuum pump nitrogen replacement step, but we found the oil easier. The vials are placed in a water bath set at 37º C for three hours, triggering polymerisation of the gel. After polymerisation, remove excess gel (now solidified) with clean gloves and lint-free wipes, revealing the specimen as a gel/tissue hybrid. Specimens were then placed directly into iodine solution.

(3) Staining

Some potential issues: This brain was very large (~140g) and did not stain throughout; fridge staining, however, may have improved this. Also, note the high density “pockets” that occur much less frequently if staining is done in the fridge.

For staining we used 1.75% IKI solution, for about 30% longer than we would normally stain—but this depends on specimens and needs to be refined a bit more. Lower-concentration solutions may yield even less shrinkage. Aki Watanabe (pers. comm.) reported slower uptake of iodine at room temperature, which he found advantageous for a more controlled stain. We also found that staining in the fridge reduced the number of high-density “pockets” of iodine producing interference during scanning.

Stain visualization and reconstruction of specimen in 3-D, using Mimics by Materialise.

Please feel free to use our protocol. If you do so, we would appreciate a citation: